DNA methylation variations underlie lettuce domestication and divergence

Background Lettuce (Lactuca sativa L.) is an economically important vegetable crop worldwide. Lettuce is believed to be domesticated from a single wild ancestor Lactuca serriola and subsequently diverged into two major morphologically distinct vegetable types: leafy lettuce and stem lettuce. However, the role of epigenetic variation in lettuce domestication and divergence remains largely unknown. Results To understand the genetic and epigenetic basis underlying lettuce domestication and divergence, we generate single-base resolution DNA methylomes from 52 Lactuca accessions, including major lettuce cultivars and wild relatives. We find a significant increase of DNA methylation during lettuce domestication and uncover abundant epigenetic variations associated with lettuce domestication and divergence. Interestingly, DNA methylation variations specifically associated with leafy and stem lettuce are related to regulation and metabolic processes, respectively, while those associated with both types are enriched in stress responses. Moreover, we reveal that domestication-induced DNA methylation changes could influence expression levels of nearby and distal genes possibly through affecting chromatin accessibility and chromatin loop. Conclusion Our study provides population epigenomic insights into crop domestication and divergence and valuable resources for further domestication for diversity and epigenetic breeding to boost crop improvement. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-024-03310-x.


Fig. S1
Fig. S1 Increased CG methylation levels during lettuce domestication.a Frequency of average methylation levels in different cytosine contexts for each cytosine.Bent arrows indicate the cutoff of methylation levels used for calculating the frequencies of average methylation levels.b A neighbor-joining phylogenetic tree of 52 Lactuca accessions based on SNPs.c,d Average CG methylation levels around genes (c) and TEs (d).Asterisks indicate significant differences (**P < 0.01, Wilcoxon signed-rank test) of CG methylation levels in cultivars as compared with the wild lettuce L. serriola (wild).

Fig. S2
Fig. S2 Phylogenetic trees based on the methylation levels of all CHG and CHH loci.a,b A neighbor-joining phylogenetic tree of 52 Lactuca accessions based on the methylation levels of all CHG (a) and CHH (b) loci.

Fig. S3
Fig. S3 Methylation levels of CHG and CHH during lettuce domestication.a, b Increased methylation levels of CHG (a) and CHH (b) in cultivated lettuces compared to the wild lettuce L. serriola (wild).Asterisks and ns indicate significant differences (**P < 0.01, twotailed paired Student's t-test) and no statistical difference (P ≥ 0.01, two-tailed paired Student's t-test) of methylation levels (mCHG and mCHH) in indicated lettuce types as compared with the wild lettuce L. serriola, respectively.

Fig. S4
Fig. S4 Identification of DMRs in lettuce cultivars.a Number of DMRs in pairwise comparisons among the wild lettuce L. serriola (wild), butterhead (butter), cos, crisp, and stem lettuce.b Distribution of high-fidelity leafy-specific DMRs in different genomic regions divided into gene body, +2 kb flanking region (2 kb upstream of TSS), -2 kb flanking region (2 kb downstream of TTS), TEs, and other intergenic regions excluding TEs.

Fig. S5
Fig. S5 Analysis of high-fidelity stem-specific DMRs.a,b Methylation levels of the highfidelity stem-specific DMRs in the wild relatives L. virosa (a) and L. indica (b).Asterisks indicate significant differences (**P < 0.01, Wilcoxon signed-rank test) as compared to mCG levels in the whole genome.c Fraction of regions with the same SNPs in stem lettuce and a wild relative (either L. saligna, L. serriola, L. virosa, or L. indica) in the global windows for DMR identification.Blue bars indicate fraction of regions with the same SNPs between stem lettuce and one wild relative, but different from other cultivars (cos, butter, and crisp lettuce) and the other wild relatives.d Distribution of the high-fidelity stemspecific DMRs in different genomic regions divided into gene body, +2 kb flanking region (2 kb upstream of TSS), and -2 kb flanking region (2 kb downstream of TTS), TEs, and other intergenic regions excluding TEs.

Fig. S6
Fig. S6 Shared domestication-induced DMRs in lettuce cultivars influence gene expression.a Distribution of shared domestication-induced DMRs in different genomic regions divided into gene body, +2 kb flanking region (2 kb upstream of TSS), -2 kb flanking region (2 kb downstream of TTS), TEs, and other intergenic regions excluding TEs.b-d Absolute expression changes in cos (b), crisp (c), and butterhead (butter) lettuce (d) relative to the wild relative L. serriola (wild) in DMR-associated proximal genes (DPGs) and distal genes (DDGs) compared with all genes.Asterisks and ns indicate significance differences (P < 0.01, Wilcoxon signed-rank test) and no statistical differences (P ≥ 0.01, Wilcoxon signedrank test), respectively.

Fig. S7
Fig. S7 Methylation levels of CG, CHG and CHH on the shared CG DMRs.a, b Methylation levels of CG, CHG and CHH on the shared hypo-DMRs (a) and shared hyper-DMRs (b).

Fig
Fig. S8 DAP-seq analysis of LsERF48.a DNA motif enriched in LsERF48-bound peaks.b Density of LsERF48-bound peaks within 10 kb of genes.Asterisk indicates a significant difference (**P < 0.01, Wilcoxon signed-rank test) between gene regions and flanking regions.c GO enrichment analysis of LsERF48 target genes.

Fig. S9
Fig. S9 Function of LsEFR48 related to ROS scavenging under salt stress.a In situ detection of ROS in N. benthamiana leaves under 150 mM or 0 mM NaCl treatment.b Quantification of relative NBT (upper panel) and DAB (lower panel) staining intensities as shown in (a) respectively.Different letters indicate significant differences (P < 0.01, two-tailed paired Student's t-test) in a pairwise comparison.

Fig. S10
Fig. S10 Chromatin accessibility of the wild lettuce L. serriola.a, b Metaplots showing chromatin accessibility of wild lettuce L. serriola on the shared domestication-induced DMRs, located near the genes (a) and far from genes (b).Asterisks indicate significant differences (**P < 0.01, Wilcoxon signed-rank test) of the chromatin accessibility in DMRs compared with the whole genome.

Fig. S11
Fig. S11 Expression changes of genes related to shared hyper-or hypo-DMRs.a, b Absolute changes for gene expression between stem lettuce and the wild lettuce L. serriola in hyper-(a) and hypo-(b) DMR-associated proximal genes (DPGs) and distal genes (DDGs) compared with all genes.Asterisks indicate significant differences (**P < 0.01, Wilcoxon signed-rank test).

Fig. S14
Fig. S14 Expression levels of genes related to shared DMRs between stem lettuce and the wild lettuce L. serriola.Asterisks and ns indicate significant difference (*P < 0.05, Wilcoxon signed-rank test) and no statistical difference (P ≥ 0.05, Wilcoxon signed-rank test), respectively.